Summary Colonic motility is the product of myogenic, neurogenic and endocrinogenic factors that regulate the excitability of smooth muscle cells (SMCs). The traditional concept of ?myogenic? has been revised to include regulatory inputs from interstitial cells (interstitial cells of Cajal (ICC) and PDGFRa+ cells). These cells are electrically coupled to SMCs, forming the SIP syncytium. While much has been learned about ICC in other organs of the GI tract, very little is known about the 4 types of ICC in colonic muscles. We developed reporter strains and mice with exclusive expression of optogenetic Ca2+ sensors in ICC. Using these mice it is possible to isolate specific types of ICC for physiological and molecular studies and to image Ca2+ transients in ICC in situ. Our preliminary data show that all ICC in the colon employ brief Ca2+ entry and release events (i.e. Ca2+ transients) to activate Ca2+-activated Cl- channels (CaCC), encoded by Ano1, in ICC. Activation of CaCC initiates inward current, and this has a depolarizing or excitatory impact on the SIP syncytium. Ca2+ transients occur spontaneously in colonic ICC, but they are also regulated by neural and hormonal inputs. Our overarching hypothesis is that Ca2+ transients in ICC are the ?myogenic? mechanism that establishes patterning of contractions in colonic muscles. Neural and hormonal modulation of ICC Ca2+ transients establish organ level mixing and propulsive motility. We will address the following questions to investigate this hypothesis: 1. What specific Ca2+ handling behaviors are manifest in the 4 classes of colonic ICC? 2. Is the behavior of ICC affected by neural and hormonal inputs and during colonic motility behaviors such as colonic migrating motor complexes (CMMC)? 3. What is the relationship between Ca2+ transients in ICC and the electrical and mechanical events that generate colonic motility? Ca2+ transients in ICC of GCaMP6f-Kit mice will be imaged in situ using confocal microscopy while recording movements and intracellular electrical activity. Preliminary data show that basal electrical and contractile patterning in colonic muscles are disrupted by ANO1 channel antagonists. These data demonstrate the key importance of ICC in colonic motor activity, because ICC express ANO1 exclusively in GI muscles. Colonic dysmotilities have been associated with reduced populations of ICC. Therefore, we will utilize animal models with reduced ICC to explore how deficiencies in these cells affect local propagating and mixing contractions and propulsive contractions, such as CMMC. Ca2+ transients will also be characterized in animals with reduced ICC to determine how loss of these cells affects spontaneous Ca2+ signaling and neural and hormonal regulation. This study will be the first comprehensive evaluation of the pacemaker activity of colonic ICC, and the first to show that ICC are responsible for developing basal motor patterning in colonic motility. Completion of the specific aims will provide understanding of why ICC loss has deleterious effects on colonic motility and provide new techniques for evaluating the heath/function of specific types of ICC and the efficacy of therapeutic agents designed to improve ICC function in colonic muscles.